Nonalcoholic steatohepatitis (NASH) is an aggressive form of nonalcoholic fatty liver disease (NAFLD) that affects 3-4% of the adult population and progresses to cirrhosis in 15-20% of affected subjects. There is currently no approved therapy for NASH and the only accurate method to diagnose this condition is a liver biopsy which is invasive, painful and occasionally associated with mortality. The lack of a point-of-care diagnostic tool to diagnose NASH, to assess disease progression versus regression and response to various therapeutics is considered by many to be the most important barrier towards large scale identification of affected individuals and for drug-development against NASH. This proposal represents a multi-PI effort to develop a lipidomics-based biomarker panel to fill these gaps in knowledge. Dr. Sanyal (PI) from the NIDDK NASH Clinical Research Network (CRN) will lead this effort and Dr. Dennis (Co-PI) who previously led the NIGMS LIPID_MAPS consortium will lead the lipid analysis. The NASH CRN data coordinating center (DCC) will continue to serve as the DCC for this proposal. The studies will draw from the NASH CRN biorepository which contains plasma drawn at the time of a liver biopsy from over 2000 highly phenotyped subjects with NAFLD. Three specific aims will test the hypothesis that changes in lipid metabolism in NAFLD allow identification of a circulating lipid signature diagnostic of NASH, disease progression and response to specific therapies. Aim 1 will define and validate a circulating lipid profile diagnostic of underlying NASH and its severity. A model building cohort and two validation cohorts will be studied. The model building cohort will include subjects with and without NAFLD. The validation cohort will include: (1) a group of subjects with varying phenotypes of NAFLD and age, gender, race and weight-matched controls without NAFLD, and (2) a prospective group of subjects in a real-world setting who are going to receive a clinically-indicated liver biopsy for suspected NASH. Aim 2 will define changes in circulating lipids diagnostic of disease progression in NASH. This will be accomplished by performing lipidomic analyses on plasma drawn at the time of baseline liver biopsies in a cohort of subjects with varying phenotypes of NAFLD who have undergone two or more liver biopsies. Aim 3 will define the circulating lipid signature associated with treatment-induced improvement of NASH. This will utilize unique sample-sets obtained during protocol-driven biopsies from subjects enrolled in clinical trials by the NASH CRN. The studies will further innovate by applying the state-of-the-art analytic platforms developed by LIPID_MAPS for diagnostics development for NASH. Preliminary data support the feasibility of the proposed studies and aims. These studies will meet all of the quality metrics for accuracy assessment of diagnostics (QUADAS) and will have a strong positive impact on the field by allowing development of a diagnostic platform that will permit point of care evaluation of NASH. This will enhance access to care and eventually improve the health of the people thereby aligning with the mission of the NIH.